Hydroxybenzohydroxamic acids, benzamides and esters as ribonucleotide reductase inhibitors

ABSTRACT

Di and trihydroxybenzohydroxamic acids, amides, alkyl substituted amides and phenyl esters, ribonucleotide reductase inhibitors.

CROSS-REFERENCE

This application is a continuation-in-part of our copending applicationSer. No. 16,472 filed Mar. 1, 1979, now U.S. Pat. No. 4,263,322, issuedApr. 21, 1981.

BACKGROUND OF THE INVENTION

There has been considerable interest in the synthesis and testing ofhydroxy-substituted benzohydroxamic acids, particularly as antimicrobialor antitumor agents. The leading research group in this area had beenheaded by Glen R. Gale. In 1966, Gale presented a paper in Proc. Soc.Exptl. Biol. Med., 122, 1236 (1966) on the selective inhibition of DNAsynthesis by salicylhydroxamic acid. He reported that cells which hadbeen preincubated with salicylhydroxamic acid for up to two hours showedonly a slight depression of RNA and protein synthesis. Gale and Hynesreported in J. Med. Chem., 11, 191 (1968) on a similar activity of otherarylhydroxamic acids including 3-hydroxy, 4-hydroxy,2,6-dihydroxy,2,3-dihydroxy, 3,5-diamino, 4-amino and3-aminobenzohydroxamic acids. The compounds were tested for theirability to inhibit DNA synthesis in Ehrlich ascites tumor cells invitro. The authors confirmed the selective inhibition of DNA synthesisby salicylhydroxamic acid. Benzohydroxamic acid was found to inhibit RNAsynthesis but the corresponding benzamide was found to be totallyinactive. 3,5-Diaminobenzohydroxamic acid and 4-aminobenzohydroxamicacid were said to display relative selectivity against DNA synthesis,but only after exposure of the cells to the agent for 1-2 hours.2,3-Dihydroxybenzohydroxamic acid was said to show a marked selectivityagainst DNA synthesis with a greater inhibition than that produced bysalicylhydroxamic acid. 3-Aminobenzohydroxamic acid was reported ascompletely inhibiting DNA synthesis but accompanied by 50 percentdepression of RNA and protein synthesis.

The authors concluded as follows: "[t]he most active and selectivecompounds were, thus, those with hydroxyl substituents in the 2 and2,3-positions on the aryl ring, while addition of the same group to the4-position yielded a virtually inactive compound. Relatively selectivebut less active compounds were those with amino groups in the 4 and3,5-positions."

Howle and Gale publishing in Proc. Soc. Exptl. Biol. Med., 131, 697(1969) [same as Chemical Abstracts 71, 57006b (1969)] describe theeffects of certain hydroxamic acids on bacterial and plant L-glutamate1-carboxylase. A few amino and hydroxy-substituted benzoylhydroxamicacids were studied. Among the new compounds reported was2,3,4-trihydroxybenzoylhydroxamic acid.

Gale, Hynes and Smith publishing in J. Med. Chem., 13, 571 (1970)described the synthesis of additional arylhydroxamic acids whichinhibited the synthesis of DNA in Ehrlich ascites tumor cells. Theauthors commented on work performed by the National Cancer Institute, N.Greenberg--Cancer Chemotherapy National Service Center, stating that4-hydroxybenzoylhydroxamic acid possessed significant antitumor activityin vivo against L1210 leukemia. The sole new compound prepared disclosedwas 2,5-dihydroxybenzoylhydroxamic acid. The most active compoundreported was 3,5-diisopropylsalicylhydroxamic acid, with anotherinteresting structure being 4-nitrobenzoylhydroxamic acid. The2,5-dihydroxybenzoylhydroxamic acid derivative was apparently of littleinterest. The authors concluded that a majority of their derivativeswhich were active in vitro were substituted in the 4-position inrelation to the hydroxamic acid group.

The most recent paper of interest by Gale and co-workers appeared inBiochemical Pharmacology, 20, 2677 (1971) [same as Chemical Abstracts,76 21107z (1972)]. This paper reported the results of testing 20arylhydroxamic acids as potential antimitogenic agents. Among thecompounds tested were the following benzoylhydroxamic acid derivatives:3-hydroxy, 4-hydroxy, 4-amino, 2,3-dihydroxy, 2,4-dihydroxy,2,5-dihydroxy, 2,6-dihydroxy, 3,5-diamino. The most active compoundstested were 4-hydroxybenzoylhydroxamic acid and the corresponding2,3-dihydroxy compound. 4-Aminobenzoylhydroxamic acid was found to beinactive as was the 2-hydroxy-4-aminobenzoyl compound. All compoundswere compared with hydroxyurea, which compound acts on susceptible cellsby inhibiting DNA synthesis through suppression of the activity ofribonucleoside diphosphate reductase. Although Gale and coworkers nevertested the effect of their compounds on ribonucleotide reductase, it wasthe conclusion of the authors that 2,3-dihydroxybenzoylhydroxamic acidprobably acted by a different mechanism. The authors also concluded thatthere was fundamental difference in action between the 4-hydroxy and the2,3-dihydroxy benzohydroxamic acids.

Other investigators have also been interested in the properties ofhydroxy-substituted benzoic acids and benzamides. Kreuchunas, U.S. Pat.No. 2,849,480, discloses a number of derivatives of2,3,6-trihydroxybenzoic acid such as the amide, the N-methylamide, theN,N-dimethylamide, etc. Utility of the compounds is said to be in thetreatment of rheumatic fever. Chemical Abstracts, 81, 120190f (1974)discloses the stabilization of aromatic amines with esters or amides ofgallic acid (3,4,5-trihydroxybenzoic acid). The primary amide and theN-methylamide are specifically disclosed. Chemical Abstracts, 85, 94115w(1978) discloses a group of 2,6-dihydroxybenzamides useful asintermediates in the preparation of the corresponding alkylcarbamoyloxyderivatives. Chemical Abstracts, 74, 112752f (1971) is anotherdisclosure of 3,5-dihydroxybenzohydroxamic acid. The compound wascondensed with meta-dihydroxybenzene and formaldehyde to provide aresin. Applicants' publications relating to the activity of di- andtrihydroxybenzohydroxamic acids, amides and esters as ribonucleotidereductase inhibitors with antineoplastic activity include papersappearing in Cancer Research, 39, 844 (1979), J. Med. Chem, 22, 589(1979), Proc. Am. Assoc. Cancer Research 18, 177 (1977), 19, 63 (1978),20, 149 (1979), Virginia Journal of Science, 29, 81 (1978), and J.Pharm. Sci., 69, 856 (1980).

3,4-Dihydroxybenzohydroxamic acid and 3,4,5-trihydroxybenzohydroxamicacid are not known. Phenolic esters of certain hydroxybenzoic acids arealso novel.

This invention provides a method of inhibiting the enzyme,ribonucleotide reductase, which comprises administering to a mammalcarrying a tumor having a relatively high ribonucleotide reductase levelan amount of a compound according to formula I below effective toinhibit ribonucleotide reductase ##STR1## wherein R¹ is NH₂, NHOH, NH(C₁-C₃)alkyl, aryl-NH, N[(C₁ -C₃)alkyl]₂ or O-phenyl; and n is 2 or 3.

Illustrative compounds useful in our novel method include the followingbenzohydroxamic acids, benzamides or phenyl hydroxybenzoates:

3,4-dihydroxybenzamide;

2,3-dihydroxybenzamide;

2,3,4-trihydroxybenzamide;

3,4,5-trihydroxybenzamide (galloamide);

2,4,5-trihydroxybenzamide;

2,3,6-trihydroxybenzamide;

phenyl 2,3-dihydroxybenzoate;

phenyl 3,4-dihydroxybenzoate;

phenyl 2,3,6-trihydroxybenzoate;

N-n-propyl 2,4,6-trihydroxybenzamide;

phenyl 3,4,5-trihydroxybenzoate (phenyl gallate);

phenyl 2,4,6-trihydroxybenzoate;

2,4,6-trihydroxybenzohydroxamic acid;

N-ethyl 3,4,5-trihydroxybenzamide.

2,3,4-trihydroxybenzohydroxamic acid;

3,4,5-trihydroxybenzohydroxamic acid;

3,4-dihydroxybenzohydroxamic acid;

2,3,5-trihydroxybenzohydroxamic acid;

2,4,5-trihydroxybenzohydroxamic acid;

2,3,6-trihydroxybenzohydroxamic acid;

phenyl-2,3,4-trihydroxybenzoate;

N-methyl 2,3,4-trihydroxybenzamide;

N-ethyl 3,4,5-trihydroxybenzamide;

N-n-propyl 3,4,5-trihydroxybenzamide;

N-phenyl 3,4-dihydroxybenzamide

and the like.

In the above formula, when R¹ is (C₁ -C₃) alkylNH or [(C₁ -C₃)alkyl]₂ N,methylamino, ethylamino, dimethylamino, n-propylamino, isopropylamino,diethylamino and the like are contemplated. The term "aryl" in "aryl-NH"includes any aromatic radical such as phenyl, thienyl, pyrimidinyl andthe like as well as an aryl radical substituted with one or morestandard substituting groups such as halo (Cl, Br, I, F) lower alkyl(methyl, ethyl, propyl), lower alkoxy (methoxy, ethoxy), nitro, cyanoetc.

This invention also provides certain novel compounds; specifically, itprovides compounds according to the above formula in which R' isO-phenyl. In addition, in a preferred aspect of this invention, thereare provided compounds in which n is 2 or 3, and two of the hydroxylgroups are at the 3 and 4 carbons of the benzene ring. A particularlypreferred group included those compounds having the above structuralfeatures in which R' is NHOH, particularly 3,4-dihydroxybenzohydroxamicacid and 3,4,5-trihydroxybenzohydroxamic acid.

The novel compounds of this invention according to formula I above inwhich R is NHOH are prepared by reacting the corresponding ester withhydroxylamine in the presence of sodium hydroxide and sodium sulfite.The following examples illustrate the preparation of hydroxamic acids.

EXAMPLE 1 Preparation of Hydroxybenzohydroxamic acids

One-half mol of sodium hydroxide as a 25% aqueous solution was addedslowly to a mixture of 0.1 mol of hydroxylamine sulfate [(NH₂ OH)₂.H₂SO₄ ] and 100 g. of ice. Next 2 g. of sodium sulfite and 0.1 mol of aparticular methyl (hydroxy substituted) benzoate were added. Thereaction was stirred at room temperature in a covered flask until theester dissolved. It was then allowed to remain overnight at 45° C. (orfor two days at ambient temperature). The reaction mixture was acidifiedwith 25% aqueous sulfuric acid with external cooling to pH=6.0.Frequently, part of the hydroxamic acid prepared in the above procedureprecipitated at this point. However, in all instances, the aqueoussolvent was evaporated under reduced pressure and the residue extractedwith hot methanol and filtered. Evaporation of the methanol from thefiltrate left the hydroxamic acid as a residue. This residue wascombined with the initial precipitate, a solution of the combinedhydroxamic acids decolorized with charcoal and the hydroxamic acidrecrystallized from hot water.

The following table gives the physical constants of severalhydroxybenzohydroxamic acids prepared by the above procedure.

                  TABLE 1                                                         ______________________________________                                        Name of Compound       M.P. °C.                                        ______________________________________                                        2,3-dihydroxybenzohydroxamic acid.sup.a                                                              218 dec                                                2,4-dihydroxybenzohydroxamic acid.sup.a                                                              178 dec                                                2,5-dihydroxybenzohydroxamic acid.sup.a                                                              216 dec                                                2,6-dihydroxybenzohydroxamic acid.sup.a                                                              215 dec                                                3,4-dihydroxybenzohydroxamic acid.sup.c                                                              174 dec                                                3,5-dihydroxybenzohydroxamic acid.sup.a                                                              230 dec                                                2,3,4-trihydroxybenzohydroxamic acid.sup.b                                                           201 dec                                                3,4,5-trihydroxybenzohydroxamic acid.sup.a                                                           230 dec                                                ______________________________________                                         .sup.a recrystallized from H.sub.2 O                                          .sup.b recrystallized from H.sub.2 O--MeOH                                    .sup.c recrystallized from ethanolethyl acetate                               dec = with decomposition                                                 

The phenyl esters (R¹ =O-phenyl) coming within the scope of formula Iare prepared from the corresponding acid and phenylchloride in thepresence of an alkali metal hydroxide. Standard esterificationprocedures (phenol plus acid) can also be used provided a large excessof phenol is employed. The preparation of these phenyl esters isillustrated by the following example:

EXAMPLE 2

A reaction mixture was prepared containing 15 g. of anhydrous gallicacid, 8 g. of phenol, 4 ml. of phosphorous oxychloride and 200 ml. ofanhydrous ether in a round-bottom flask. The mixture was heated torefluxing temperature for about 18 hours. The ether solvent was removedby evaporation. Water was added to the resulting residue and the pH ofthe aqueous layer adjusted to about 5 by the addition of dilute aqueoussodium hydroxide. Phenyl gallate, being insoluble at pH=5, separated andwas extracted with ether. The ether extracts were separated and driedand the ether removed therefrom by evaporation. Recrystallization of theresidue yielded phenyl gallate melting at about 190°-191° C.

Analysis Calculated: C, 63.41; H, 4.09. Found: C, 62.69; H, 4.14.

Preparation of starting materials

The di and trihydroxybenzoic acid esters employed as starting materialsin most of the above syntheses were themselves prepared from thecorresponding di or trihydroxybenzoic acid according to the followingprocedure: one-tenth mol of the di or trihydroxybenzoic acid wasrefluxed for about 24 hours with 100 ml. of methanol containing 2% (v/v)concentrated sulfuric acid. At this time, the excess methanol wasremoved by evaporation under reduced pressure and the residual esterssuspended in water. The esters were isolated therefrom by publishedprocedures. The following di or trihydroxybenzoic acid methylesters areknown: 2,3-dihydroxy, 2,5-dihydroxy, 3,4-dihydroxy, 3,4,5-dihydroxy.

2,3,4-Trihydroxybenzoic acid was not commercially available, but wasreadily prepared by heating pyrogallol with sodium bicarbonate (20 g. to30 g.) in a mixture of 30 ml. of water and 20 ml. of mesitylene.Acidification of the resulting suspension with concentrated aqueoushydrochloric acid yielded 2,3,4-trihydroxybenzoic acid melting at about220° C. with decomposition, (literature melting point=221° C.) Methyl2,3,4-trihydroxybenzoate was prepared by heating the acid with amethanol-sulfuric acid mixture. The compound thus prepared melted at151° C. (compared to a literature melting point of 153° C.). The methylester of 2,6-dihydroxybenzoic acid was synthesized by preparing thesilver salt of the acid and treating it with methyl iodide. Methyl2,6-dihydroxybenzoic melted at 67° C., (published melting point=67°-68°C.).

As stated above, the compounds represented by formula I above have theability to inhibit ribonucleotide reductase, an enzyme involved in thereductive conversion of ribonucleotides to deoxyribonucleotides. Thisenzymatic reaction is a rate controlling step in the biosyntheticpathway leading to DNA and cell replication. In general, theribonucleotide reductase level is closely correlated with cellularreplication. Thus, it is not surprising that the compounds of thisinvention, which are potent ribonucleotide reductase inhibitors, arealso capable of prolonging the life of mice carrying transplanted tumorssince replication of tumor cells is equally inhibited. In particular, wehave found that administration of a compound of this inventionrepresented by formula I above prolongs the life of mice inoculated withL1210 leukemia, a tumor not ordinarily susceptible to chemotherapy. Inaddition, the compounds have demonstrated activity against othertransplanted tumors; i.e., Lewis Lung, X-5563 lymphoma and 755adenocarcinoma, B₁₆ melanoma, C-8 colon, breast and human xenographs.

The results of several biological tests of compound according to formulaI are incorporated in the following tables.

Table 2 gives ribonucleotide reductase data for certain compounds offormula I. In the table, column 1 gives the substitution pattern in thebenzene ring, column 2, the R¹ group, column 3, the ID₅₀ (inhibitorydose in micromolar concentration which inhibits ribonucleotide reductaseby 50%), column 4, the average percent (± standard error) increase inthe life span (ILS) of 8 treated mice inoculated with L1210 leukemiacompared with 8 control mice also inoculated with L1210. The daily doselevel of the drug which was administered for 8 days is indicated incolumn 5. Table 3 gives the relative potency of the compoundsrepresented by formula I above as inhibitors of ribonucleotide reductasecompared to hydroxyurea whose potency was given an arbitrary value of1.0. In table 3, column 1 gives the name of the compound and column 2the relative inhibitory potency of each of the compounds of column 1.

                  TABLE 2                                                         ______________________________________                                         ##STR2##                                                                     (OH)   R.sup.1  ID.sub.50 (μM)                                                                        ILS.    Dose mg/kg                                 ______________________________________                                        2,3    NHOH     8          36 ± 11                                                                            200                                        2,4    NHOH     250        42 ± 7                                                                             500                                        2,5    NHOH     200        30 ± 14                                                                            300                                        2,6    NHOH     100        23 ± 11                                                                             88                                        3,4    NHOH     30         103 ± 15                                                                           600                                        3,5    NHOH     400        52 ± 22                                                                            1000                                       2,3,4  NHOH     3.5        30 ± 7                                                                             125                                        3,4,5  NHOH     10         53 ± 19                                                                            400                                        3,4    NH.sub.2 50         35 ± 11                                                                            1200                                       2,3,4  NH.sub.2 5          48 ± 7                                                                             200                                        3,4,5  NH.sub.2 10         42 ± 5                                                                             250                                        3,4,5  NHCH.sub.3                                                                             25         60 ± 29                                                                            500                                        ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                            Inhibitory Potency                                                            for ribonucleotide                                        Name of compound    reductase                                                 ______________________________________                                        Hydroxyurea (control compound)                                                                    1.0                                                       3,5-dihydrobenzohydroxamic acid                                                                   1.2                                                       2,4-dihydroxybenzohydroxamic acid                                                                 2.0                                                       2,5-dihydroxybenzohydroxamic acid                                                                 2.5                                                       2,6-dihydroxybenzohydroxamic acid                                                                 5.0                                                       Vicinal hydroxyls                                                             2,3-dihydroxybenzohydroxamic acid                                                                 63                                                        3,4-dihydroxybenzohydroxamic acid                                                                 17                                                        3,4-dihydroxybenzamide                                                                            10                                                        3,4,5-trihydroxybenzohydroxamic acid                                                              50                                                        2,3,4-trihydroxybenzohydroxamic acid                                                              140                                                       3,4,5-trihydroxybenzamide                                                                         50                                                        2,3,4-trihydroxybenzamide                                                                         100                                                       phenyl 3,4,5-trihydroxybenzoate                                                                   33                                                        ______________________________________                                    

In the above determination of ID₅₀ 's in Table 3, ribonucleotidereductase is partially purified from Novikoff hepatoma by a proceduresimilar to that set forth by Elford et al. J. Biol. Chem. 245, 5228(1970). The activity of the enzyme was measured by a slightly modifiedassay procedure originally developed by Reichard et al. id, 236, 1150(1969). This procedure measures the conversion of CDP to dCDP. The assaymixture (0.34 ml.) contains 3μ Ci of [³ H] CDP (specific activity 14-19Ci/μmol), 3.3μ mole ATP, 5.9μ moles magnesium chloride, 8.8μ moles Hepesbuffer at pH=7.5, 15μ moles dithiothreitol and enzyme protein between0.4 and 1.3 mg. Incubation was provided for forty minutes at 30° C. Ionexchange chromatography employing Dowex 50 (H⁺) resin is used toseparate the product from the substrate. The inhibitors were dissolvedin water and a mixture of water and up to 1% ethanol or 2%dimethylsulfoxide, neither one of which inhibited the enzyme at theseconcentrations. Each inhibitor was tested at a minimum of threeconcentrations and the active compounds reassayed at least oneadditional time. ID₅₀ 's in μmoles were estimated from graphssummarizing results for each compound.

In addition, positive experimental results obtained by testing compoundsrepresented by formula I against five transplanted tumors in mice areset forth in Tables 4-8. The L1210 data (Table 4) are in addition to thedata in Table 2. All compounds were administered once or twice daily for9-10 days starting 24 hours after inoculation with the tumor. In tables4-8, column 1 gives the name of the compound, column 2, the dose level,and column 3 percent increase in survival time or prolongation of life.

                  TABLE 4                                                         ______________________________________                                        Activity vs. L-1210 Leukemia                                                                       dose in % increase in                                    name of compound     mg/kg   survival time                                    ______________________________________                                        galloamide           250     toxic                                                                 225     10                                                                    200     46, 108                                                               175     55                                                                    150     50                                               gallohydroxamic acid 120     54                                                                    100     58, 55                                                                 75     60                                               3,4-dihydroxybenzohydroxamic acid                                                                  225     65                                                                    200     54, 141                                                                       (4 survivors)                                                         175     toxic                                            2,3,4-trihydroxybenzohydroxamic acid                                                               25-50   17-21                                            phenyl gallate       200     56                                               hydroxyurea (positive control)                                                                      71     58                                                                     60     92                                                                     40     27                                                                     30     27                                               ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Activity vs. Lewis Lung                                                                                       % tumor                                       name of compound    dose in mg/kg                                                                             inhibition                                    ______________________________________                                        gallohydroxamic acid                                                                              100         40                                            galloamide          200         21                                            3,4-dihydroxybenzohydroxamic acid                                                                 200         14                                                                400         31-58                                         2,3,4-trihydroxybenzohydroxamic acid                                                              25-50       13-14                                         phenyl gallate       50          2                                                                200         14                                            hydroxyurea (positive control)                                                                     71         27                                                                400         44                                            ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Activity vs. L-5563 lymphoma                                                                                  %                                             Name of compound    dose in mg/kg                                                                             inhibition                                    ______________________________________                                        gallohydroxamic acid                                                                              100         41                                            galloamide          200         toxic                                         3,4-dihydroxybenzohydroxamic acid                                                                 200         24                                            2,3,4-trihydroxybenzohydroxamic acid                                                              25-50       toxic                                         phenylgallate       200         19                                            hydroxyurea (positive control)                                                                     71         toxic                                         ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Activity vs. CA-755 adenocarcinoma                                                                           %                                              Name of compound    Dose mg/kg inhibition                                     ______________________________________                                        gallohydroxamic acid                                                                              100        28                                                                 150        11                                                                 175        29                                             galloamide          200        38                                             3,4-dihydroxybenzohydroxamic acid                                                                 100        16                                                                 200        25, 36                                                             300        52, toxic                                      2,3,4-trihydroxybenzohydroxamic acid                                                              25-50      12-18                                          phenyl gallate      100        27                                                                 200        11                                             hydroxyurea (positive control)                                                                     71        27                                                                 200        26                                             ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Activity vs. P388 Leukemia                                                    Name of Compound                                                                             Dose in mg/kg                                                                             % Prolongation                                     ______________________________________                                        3,4,5-Trihydroxybenzo-                                                                       225         79%                                                hydroxamic acid                                                                              200         62%                                                3,4-dihydroxybenzo-                                                                          225         89%                                                hydroxamic acid                                                                              200         51%                                                galloamide     225         55%                                                               200         40%                                                               175         43%                                                ______________________________________                                    

In addition, the compound 3,4-dihydroxybenzenehydroxamic acid was activeagainst CX-1 colon xenograft using the following protocol:

0% tumor inhibition at 800 mg/kg

dose level every fourth day,

14% at 400 mg/kg level and

48% at 200 mg/kg level.

3,4,5-trihydroxybenzohydroxamic acid gave the following results againstthe same tumor using the same protocol.

44% inhibition at 800 mg/kg

40% inhibition at 400 mg/kg

17% inhibition at 200 mg/kg

The same drug showed excellent prolongation of life for mice inoculatedwith P-388 leukemia: 23-53% at doses ranging from 50-400 mg/kg everyfifth day. The same two compounds were effective in inhibiting thegrowth of the following transplanted tumors--B₁₆ melanoma, M-1 humanbreast xenograft, LX-1 human lung xenograft, CD8F mammary tumor andcolon 38 tumor--as follows:

    ______________________________________                                        3,4-dihydroxybenzohydroxamic acid                                             ______________________________________                                        CD8F,   300 mg/kg  52%       inhibition                                               600 mg/kg  11%       inhibition                                       C8      400 mg/kg  52%       inhibition                                               200 mg/kg  15%       inhibition                                               100 mg/kg  8%        inhibition                                               50 mg/kg   12%       inhibition                                       B16     400 mg/kg  25%       increase in survival                                     200 mg/kg  24-25%    increase in survival                             LX-1    800 mg/kg  27%       inhibition                                               400 mg/kg  26%       inhibition                                               200 mg/kg  0%        inhibition                                       ______________________________________                                        3,4,5-trihydroxybenzohydroxamic acid                                          ______________________________________                                        CD8F    600 mg/kg  22%       inhibition                                               300 mg/kg  0%        inhibition                                       C8      800 mg/kg  60%       inhibition                                               400 mg/kg  17%       inhibition                                               200 mg/kg  32%       inhibition                                               100 mg/kg  30%       inhibition                                               50 mg/kg   20%       inhibition                                       B16     200 mg/kg  18%       increase in survival                                     100 mg/kg  14%       increase in survival                             M-1     1600 mg/kg 41%       inhibition                                               800 mg/kg  56%       inhibition                                               400 mg/kg  21%       inhibition                                               200 mg/kg  69%       inhibition                                       ______________________________________                                    

In a series of studies against L-1210 leukemia, in addition to thosestudies set forth in Table 2 and 4, 3,4-dihydroxybenzohydroxamic acidgave the following increases in survival times at the dose levelsspecified. Drug was administered daily for nine days.

    ______________________________________                                        Dose mg/kg   % increase in survival                                           ______________________________________                                        600          54, 64                                                           480          106, 87, 67                                                      400          67                                                               384          62, 85                                                           336          55                                                               307          67, 76                                                           200          67                                                               245          45, 68                                                           235          39                                                               196          52                                                               164          21                                                               100          37                                                                50          25                                                                25          15                                                               ______________________________________                                    

Similar data for 3,4,5-trihydroxybenzohydroxamic acid are as follows:

    ______________________________________                                                    % increase in                                                            Dose survival time                                                     ______________________________________                                               800  16                                                                       400  0, 4                                                                     200  54, 56                                                                   100  17, 39                                                                    50  27, 29                                                            ______________________________________                                    

    ______________________________________                                        2,3,4-trihydroxybenzohydroxamic acid                                                         % increase in                                                  Dose mg/kg     survival time                                                  ______________________________________                                        200            Toxic                                                          100            16                                                              50             8                                                              25            10                                                             ______________________________________                                    

The above data clearly show the enhanced anti-tumor activity oftrihydroxybenzohydroxamic acid where the hydroxy groups are vicinal butno hydroxyl is ortho. Similar data are available for the vicinaldihydroxybenzohydroxamic acids with and without an ortho-hydroxysubstituent in an L-1210 leukemic test in mice as follows:

    ______________________________________                                        3,4-dihydroxybenzohydroxamic acid                                                            % increase in                                                  Dose mg/kg     survival time                                                  ______________________________________                                        800            Toxic                                                          643            65                                                             600            94, 103                                                        543            77                                                             500            84                                                             453            125                                                            300            56                                                             100            25                                                             ______________________________________                                    

    ______________________________________                                        2,3-dihydroxybenzohydroxamic acid                                                            % increase in                                                  Dose mg/kg     survival time                                                  ______________________________________                                        500            Toxic                                                          450            about 0                                                        300            about 0, toxic                                                 200            6, 36                                                          ______________________________________                                    

In a direct side-by-side comparison with L-1210 leukemia, the followingresults were obtained.

    ______________________________________                                        3,4-dihydroxybenzohydroxamic acid                                                            % increase in                                                  Dose mg/kg     survival time                                                  ______________________________________                                        500            70                                                             250            32                                                             ______________________________________                                    

    ______________________________________                                        2,3-dihydroxybenzohydroxamic acid                                                            % increase in                                                  Dose mg/kg     survival time                                                  ______________________________________                                        250            16                                                             125             0                                                             ______________________________________                                    

The superiority as anti-leukemic drugs of the 3,4 and 3,4,5hydroxysubstitution patterns in the novel benzhydroxamic derivatives ofthis invention compared with the 2,3 and 2,3,4 hydroxy substitutionpatterns of the prior art compounds is clearly established by the abovedata.

We claim:
 1. A method of inhibiting ribonucleotide reductase whichcomprises administering to a mammal carrying a tumor having a relativelyhigh ribonucleotide reductase level, an amount of a compound accordingto the following formula effective to inhibit ribonucleotide reductase##STR3## wherein Z and Z' are individually H or OH, at least one of Zand Z' being OH, and R¹ is NHOH or O-phenyl.
 2. A method according toclaim 1 in which 3,4-dihydroxybenzohydroxamic acid is administered.
 3. Amethod according to claim 1 in which 3,4,5-trihydroxybenzohydroxamicacid is administered.
 4. A method according to claim 1 in which phenylgallate is administered.
 5. A method according to claim 1 in which Z andZ' are individually H or OH, at least one of Z and Z' being OH, and R¹is O-phenyl.
 6. A compound of the formula ##STR4## wherein n is 2 or 3.7. A compound according to claim 6, said compound being phenyl gallate.8. A compound of the formula ##STR5## wherein Z is H or OH.
 9. Acompound according to claim 8, said compound being3,4-dihydroxybenzohydroxamic acid.
 10. A compound according to claim 8,said compound being 3,4,5-trihydroxybenzohydroxamic acid.